Project Summary Hearing loss is a potentially debilitating condition that afflicts to varying extents more than 30 million individuals in the United States. Commonly caused by destruction of the mechanosensory hair cells in the inner ear, most hearing loss in humans is permanent because these cells are not naturally replenished. Unlike those in the human ear, hair cells of non-mammalian vertebrates including fishes, amphibians, and reptiles including birds can regenerate throughout life. Despite extensive research, the molecular and cellular bases of this difference are not yet well understood. Because a more complete comprehension of hair-cell regeneration in non-mammalian model systems will foster the development of regenerative therapies for hearing loss, our current research focuses on elucidating the mechanism of hair-cell regeneration in one such system, the zebrafish lateral line. The lateral line comprises an array of superficially located hair cell-containing organs called neuromasts that detect water displacement. We have devised procedures for transgenically labeling and isolating the putative progenitors?called mantle cells?that are thought to give rise to hair cells in neuromasts during regeneration. Microarray analysis has revealed numerous genes that are highly and specifically expressed in mantle cells, as well as genes whose expression in these cells is modulated in response to ototoxic insult. The resulting data set places us in a unique position to determine which molecular pathways activate or repress the proliferation of progenitors and the subsequent replacement of hair cells. Among the top candidate genes for controlling progenitor proliferation are those encoding the transmembrane protein Tspan1 and the protocadherins Fat1a and Fat1b, which are highly enriched in mantle cells and are down- regulated during regeneration. We propose to use loss-of-function (antisense-mediated knockdown and existing mutant lines) as well as gain-of-function (mRNA-mediated overexpression and misexpression by means of an inducible transgenic system) to test the functions of Tspan1, Fat1a, and Fat1b in hair-cell development and regeneration. Through these studies we hope to uncover previously unappreciated molecular mechanisms governing hair-cell regeneration, which may contribute to the future development of therapies for hearing loss.